This application describes a multifunctional microscope system capable of patterned excitation of various optically sensitive targets. This custom-designed instrument will be used to explore cutting edge questions in neural cell biology, single neuron physiology, and microcircuit function. It will incorporate a pulsed-IR source for PMT-based two-photon imaging;in addition it will have another optical path allowing for patterned, single-photon illumination at various wavelengths (405, 473 and 594 nm). Patterned illumination will be accomplished with spatial light modulator (SLM) technology which permits user defined patterns of illumination to be delivered to the specimen plane (Lutz et al., 2008;Nikolenko et al., 2008). A high speed CCD camera will be used as an alternative detector for experiments in which high time resolution (i.e.>1 kHz) optical measurements are necessary. This will allow optical measurements of electrical activity from many single neurons labeled with a novel voltage sensor developed by one of the group members. A survey of existing microscopy capabilities at UCLA demonstrates that no equipment with these capabilities is available;indeed two of the users have collaborations with overseas laboratories to enable the work. This instrument should allow experimental manipulation of several cutting edge optical tools with unparalleled spatial and temporal precision. These include: 1) photoactivatable or photoconvertible proteins (PA-GFP, Dendra2, DRONPA) useful for cell biological studies of nervous system function, 2) caged neurotransmitters that can be used to photostimulate individual neurons or circuits, 3) photosensitive channels (channel- and halorhodopsin) or reversible photo switches (PALs) to activate or inactivate specific circuit elements, and 4) a novel, ultra rapid optical reporter of membrane potential that enables simultaneous measurements of neural activity from many individual neurons. The application details how the instrument system will directly benefit more than 10 currently funded NIH projects on which the five major users serve as Principal Investigators. Research in these laboratories is directed at understanding fundamental issues in neurophysiology and neural cell biology including the molecular mechanisms involved in excitation-contraction coupling (Vergara), exocytosis (Schweizer), and neuronal plasticity (Martin) as well as aspects of microcircuit function in brainstem respiratory centers (Feldman), vestibular epithelium (Schweizer), and in cerebellar cortex (Otis). Various UCLA Departments and the UCLA School of Medicine will provide $92,922 in funding to enable the purchase of the system;they will also contribute more than $46,000 per year in ongoing funding to cover the service contract for the equipment. In addition, the user group will provide approximately $90,000 of electrophysiological, micro perfusion, and temperature control equipment so that neurophysiological experiments can be done in parallel with optical measurements under controlled physiological conditions. These pledges totaling well over $250,000 in institutional support are evidence of the scientific enthusiasm and intense need for this type of instrumentation at UCLA. PUBLIC HEALTH RELEVANCE: This grant would provide funding for a state-of-the-art microscope system incorporating advanced optical technology that enables parallel manipulation and/or measurement of neuronal activity within various brain microcircuits. The system will also permit sophisticated tracking and manipulation of optically-tagged signaling proteins with unprecedented precision in cell biological experiments. UCLA has a large and highly collaborative neuroscience research community and this equipment will accelerate progress in the laboratories of the NIH-funded major users whose research on basic mechanisms underlying disorders of breathing, movement, balance, and learning is currently funded by more than 15 PHS grants.